Drug abuse is a prevalent psychiatric disorder with immense negative public health consequences. As a result, understanding its neural underpinnings is a focus of intense research. The dopamine (DA) transporter (DAT) is a primary site of action of drugs of abuse such as amphetamine and is critical in regulating DA neurotransmission by high affinity transport of DA into the terminal. Understanding how the DAT is regulated is therefore of fundamental importance to studies of amphetamine abuse. In this regard, there is converging evidence that insulin can produce profound regulatory control of DAT activity. It is known for example that both food restriction as well as experimentally-induced diabetes have major effects on behavioral responses to amphetamine. The significance of these observations is underlined by the high co-morbidity of eating disorders and drug abuse. Recently it has been reported that rates of DA uptake are decreased in hypoinsulinemic rats and that insulin applied intracerebroventricularly to rats or to cells stably transfected with the DAT increases DA uptake. Importantly, it seems that insulin can interfere with the action of amphetamine at the DAT and can prevent amphetamine-induced internalization of the DAT. Of particular importance to the present proposal is that diabetic rats with a history of amphetamine self-administration show increased rather than decreased DA uptake. Insulin and its signaling pathways may therefore represent a novel target for the development of new treatments for drug abuse. To date no studies have assessed the role of insulin in controlling DAT activity in vivo. Because of the potential impact of insulin in promoting the abuse potential of drugs the proposed study will use an innovative approach, high-speed chronoamperometry, to measure insulin-dependent changes in DA efflux and clearance in vivo. Because a major signaling mechanism that underlies insulin's cellular actions is stimulation of phosphatidylinositol (PI)-3 kinase these studies will also test the hypothesis that DA effiux and clearance will be increased and decreased respectively, upon activation and inhibition of PI-3 kinase. Importantly, these studies will determine the relationship between the action of amphetamine and insulin status as they relate to DAT activity. Our general hypothesis is that the kinetics of DA efflux and clearance as well the locomotor stimulatory effects of amphetamine will be tightly correlated with insulin status. Restoration of insulin to normal levels will restore normal responses. The results obtained here will not only improve our fundamental understanding of DAT regulation but importantly the neural circuitry controlling reward and motivation, as well as provide a framework for a larger RO1 application. In turn, these studies may help to illuminate the neural mechanisms underlying the high co-morbidity of eating disorders and drug abuse.